TMEM16A expression in cholinergic neurons of the medial habenula mediates anxiety-related behaviors.

TMEM16A, a Ca2+ -activated Cl- channel, is known to modulate the excitability of various types of cells; however, its function in central neurons is largely unknown. Here, we show the specific expression of TMEM16A in the medial habenula (mHb) via RNAscope in situ hybridization, immunohistochemistry, and electrophysiology. When TMEM16A is ablated in the mHb cholinergic neurons (TMEM16A cKO mice), the slope of after-hyperpolarization of spontaneous action potentials decreases and the firing frequency is reduced. Reduced mHb activity also decreases the activity of the interpeduncular nucleus (IPN). Moreover, TMEM16A cKO mice display anxiogenic behaviors and deficits in social interaction without despair-like phenotypes or cognitive dysfunctions. Finally, chemogenetic inhibition of mHb cholinergic neurons using the DREADD (Designer Receptors Exclusively Activated by Designer Drugs) approach reveals similar behavioral phenotypes to those of TMEM16A cKO mice. We conclude that TMEM16A plays a key role in anxiety-related behaviors regulated by mHb cholinergic neurons and could be a potential therapeutic target against anxiety-related disorders.

ANO1/TMEM16A regulates process maturation in radial glial cells in the developing brain.

Neural stem cells (NSCs) are primary progenitor cells in the early developmental stage in the brain that initiate a diverse lineage of differentiated neurons and glia. Radial glial cells (RGCs), a type of neural stem cell in the ventricular zone, are essential for nurturing and delivering new immature neurons to the appropriate cortical target layers. Here we report that Anoctamin 1 (ANO1)/TMEM16A, a Ca2+-activated chloride channel, mediates the Ca2+-dependent process extension of RGCs. ANO1 is highly expressed and functionally active in RGCs of the mouse embryonic ventricular zone. Knockdown of ANO1 suppresses RGC process extension and protrusions, whereas ANO1 overexpression stimulates process extension. Among various trophic factors, brain-derived neurotrophic factor (BDNF) activates ANO1, which is required for BDNF-induced process extension in RGCs. More importantly, Ano1-deficient mice exhibited disrupted cortical layers and reduced cortical thickness. We thus conclude that the regulation of RGC process extension by ANO1 contributes to the normal formation of mouse embryonic brain.

Anoctamin 1 (TMEM16A) is essential for testosterone-induced prostate hyperplasia.

Benign prostatic hyperplasia (BPH) is characterized by an enlargement of the prostate, causing lower urinary tract symptoms in elderly men worldwide. However, the molecular mechanism underlying the pathogenesis of BPH is unclear. Anoctamin1 (ANO1) encodes a Ca(2+)-activated chloride channel (CaCC) that mediates various physiological functions. Here, we demonstrate that it is essential for testosterone-induced BPH. ANO1 was highly amplified in dihydrotestosterone (DHT)-treated prostate epithelial cells, whereas the selective knockdown of ANO1 inhibited DHT-induced cell proliferation. Three androgen-response elements were found in the ANO1 promoter region, which is relevant for the DHT-dependent induction of ANO1. Administration of the ANO1 blocker or Ano1 small interfering RNA, inhibited prostate enlargement and reduced histological abnormalities in vivo. We therefore concluded that ANO1 is essential for the development of prostate hyperplasia and is a potential target for the treatment of BPH.

Conditional genetic deletion of Ano1 in interstitial cells of Cajal impairs Ca2+ transients and slow waves in adult mouse small intestine.

Myenteric plexus interstitial cells of Cajal (ICC-MY) in the small intestine are Kit+ electrical pacemakers that express the Ano1/TMEM16A Ca2+-activated Cl- channel, whose functions in the gastrointestinal tract remain incompletely understood. In this study, an inducible Cre-LoxP-based approach was used to advance the understanding of Ano1 in ICC-MY of adult mouse small intestine. KitCreERT2/+;Ano1Fl/Fl mice were treated with tamoxifen or vehicle, and small intestines (mucosa free) were examined. Quantitative RT-PCR demonstrated ~50% reduction in Ano1 mRNA in intestines of conditional knockouts (cKOs) compared with vehicle-treated controls. Whole mount immunohistochemistry showed a mosaic/patchy pattern loss of Ano1 protein in ICC networks. Ca2+ transients in ICC-MY network of cKOs displayed reduced duration compared with highly synchronized controls and showed synchronized and desynchronized profiles. When matched, the rank order for Ano1 expression in Ca2+ signal imaged fields of view was as follows: vehicle controls>>>cKO(synchronized)>cKO(desynchronized). Maintenance of Ca2+ transients' synchronicity despite high loss of Ano1 indicates a large functional reserve of Ano1 in the ICC-MY network. Slow waves in cKOs displayed reduced duration and increased inter-slow-wave interval and occurred in regular- and irregular-amplitude oscillating patterns. The latter activity suggested ongoing interaction by independent interacting oscillators. Lack of slow waves and depolarization, previously reported for neonatal constitutive knockouts, were also seen. In summary, Ano1 in adults regulates gastrointestinal function by determining Ca2+ transients and electrical activity depending on the level of Ano1 expression. Partial Ano1 loss results in Ca2+ transients and slow waves displaying reduced duration, while complete and widespread absence of Ano1 in ICC-MY causes lack of slow wave and desynchronized Ca2+ transients.NEW & NOTEWORTHY The Ca2+-activated Cl- channel, Ano1, in interstitial cells of Cajal (ICC) is necessary for normal gastrointestinal motility. We knocked out Ano1 to varying degrees in ICC of adult mice. Partial knockout of Ano1 shortened the widths of electrical slow waves and Ca2+ transients in myenteric ICC but Ca2+ transient synchronicity was preserved. Near-complete knockout was necessary for transient desynchronization and loss of slow waves, indicating a large functional reserve of Ano1 in ICC.

TRPM2, a Susceptibility Gene for Bipolar Disorder, Regulates Glycogen Synthase Kinase-3 Activity in the Brain.

Bipolar disorder (BD) is a psychiatric disease that causes mood swings between manic and depressed states. Although genetic linkage studies have shown an association between BD and TRPM2, a Ca(2+)-permeable cation channel, the nature of this association is unknown. Here, we show that D543E, a mutation of Trpm2 that is frequently found in BD patients, induces loss of function. Trpm2-deficient mice exhibited BD-related behavior such as increased anxiety and decreased social responses, along with disrupted EEG functional connectivity. Moreover, the administration of amphetamine in wild-type mice evoked a notable increase in open-field activity that was reversed by the administration of lithium. However, the anti-manic action of lithium was not observed in the Trpm2(-/-) mice. The brains of Trpm2(-/-) mice showed a marked increase in phosphorylated glycogen synthase kinase-3 (GSK-3), a key element in BD-like behavior and a target of lithium. In contrast, activation of TRPM2 induced the dephosphorylation of GSK-3 via calcineurin, a Ca(2+)-dependent phosphatase. Importantly, the overexpression of the D543E mutant failed to induce the dephosphorylation of GSK-3. Therefore, we conclude that the genetic dysfunction of Trpm2 causes uncontrolled phosphorylation of GSK-3, which may lead to the pathology of BD. Our findings explain the long-sought etiologic mechanism underlying the genetic link between Trpm2 mutation and BD. SIGNIFICANCE STATEMENT: Bipolar disorder (BD) is a mental disorder that causes changes in mood and the etiology is still unknown. TRPM2 is highly associated with BD; however, its involvement in the etiology of BD is still unknown. We show here that TRPM2 plays a central role in causing the pathology of BD. We found that D543E, a mutation of Trpm2 frequently found in BD patients, induces the loss of function. Trpm2-deficient mice exhibited mood disturbances and impairments in social cognition. TRPM2 actively regulates the phosphorylation of GSK-3, which is a main target of lithium, a primary medicine for treating BD. Therefore, abnormal regulation of GSK-3 by hypoactive TRPM2 mutants accounts for the pathology of BD, providing the possible link between BD and TRPM2.

Cellular functions of TMEM16/anoctamin.

Ca(2+)-activated Cl(-) channels (CaCCs) are a class of Cl(-) channels activated by intracellular Ca(2+) that are known to mediate numerous physiological functions. In 2008, the molecular identity of CaCCs was found to be anoctamin 1 (ANO1/TMEM16A). Its roles have been studied in electrophysiological, histological, and genetic aspects. ANO1 is known to mediate Cl(-) secretion in secretory epithelia such as airways, salivary glands, intestines, renal tubules, and sweat glands. ANO1 is a heat sensor activated by noxious heat in somatosensory neurons and mediates acute pain sensation as well as chronic pain. ANO1 is also observed in vascular as well as airway smooth muscles, controlling vascular tone as well as airway hypersensitivity. ANO1 is upregulated in numerous types of cancers and thus thought to be involved in tumorigenesis. ANO1 is also found in proliferating cells. In addition to ANO1, involvement of its paralogs in pathophysiological conditions was also reported. ANO2 is involved in olfaction, whereas ANO6 works as a scramblase whose mutation causes a rare bleeding disorder, the Scott syndrome. ANO5 is associated with muscle and bone diseases. Recently, an X-ray crystal structure of a fungal TMEM16 was reported, which explains a precise molecular gating mechanism as well as ion conduction or phospholipid transport across the plasma membrane.

Dynamic modulation of ANO1/TMEM16A HCO3(-) permeability by Ca2+/calmodulin.

Anoctamin 1 (ANO1)/transmembrane protein 16A (TMEM16A) is a calcium-activated anion channel that may play a role in HCO(3)(-) secretion in epithelial cells. Here, we report that the anion selectivity of ANO1 is dynamically regulated by the Ca(2+)/calmodulin complex. Whole-cell current measurements in HEK 293T cells indicated that ANO1 becomes highly permeable to HCO(3)(-) at high [Ca(2+)](i). Interestingly, this result was not observed in excised patches, indicating the involvement of cytosolic factors in this process. Further studies revealed that the direct association between ANO1 and calmodulin at high [Ca(2+)](i) is responsible for changes in anion permeability. Calmodulin physically interacted with ANO1 in a [Ca(2+)](i)-dependent manner, and addition of recombinant calmodulin to the cytosolic side of excised patches reversibly increased P(HCO3)/P(Cl). In addition, the high [Ca(2+)](i)-induced increase in HCO(3)(-) permeability was reproduced in mouse submandibular gland acinar cells, in which ANO1 plays a critical role in fluid secretion. These results indicate that the HCO(3)(-) permeability of ANO1 can be dynamically modulated and that ANO1 may play an important role in cellular HCO(3)(-) transport, especially in transepithelial HCO(3)(-) secretion.

Two helices in the third intracellular loop determine anoctamin 1 (TMEM16A) activation by calcium.

Anoctamin 1 (ANO1)/TMEM16A is a Cl(-) channel activated by intracellular Ca(2+) mediating numerous physiological functions. However, little is known of the ANO1 activation mechanism by Ca(2+). Here, we demonstrate that two helices, "reference" and "Ca(2+) sensor" helices in the third intracellular loop face each other with opposite charges. The two helices interact directly in a Ca(2+)-dependent manner. Positively and negatively charged residues in the two helices are essential for Ca(2+)-dependent activation because neutralization of these charges change the Ca(2+) sensitivity. We now predict that the Ca(2+) sensor helix attaches to the reference helix in the resting state, and as intracellular Ca(2+) rises, Ca(2+) acts on the sensor helix, which repels it from the reference helix. This Ca(2+)-dependent push-pull conformational change would be a key electromechanical movement for gating the ANO1 channel. Because chemical activation of ANO1 is viewed as an alternative means of rescuing cystic fibrosis, understanding its gating mechanism would be useful in developing novel treatments for cystic fibrosis.

TRPM2 mediates the lysophosphatidic acid-induced neurite retraction in the developing brain.

Intracellular Ca(2+) signal is a key regulator of axonal growth during brain development. As transient receptor potential (TRP) channels are permeable to Ca(2+) and mediate numerous brain functions, it is conceivable that many TRP channels would regulate neuronal differentiation. We therefore screened TRP channels that are involved in the regulation of neurite growth. Among the TRP channels, the Trpm2 level was inversely associated with neurite growth. TRPM2 was highly expressed in embryonic brain. Pharmacological perturbation or knockdown of TRPM2 markedly increased the axonal growth, whereas its overexpression inhibited the axonal growth. Addition of ADP ribose, an endogenous activator of TRPM2, to PC12 cells significantly repressed the axonal growth. TRPM2 was actively involved in the neuronal retraction induced by cerebrospinal fluid-rich lysophosphatidic acid (LPA). More importantly, neurons isolated from the brain of Trpm2-deficient mice have significantly longer neurites with a greater number of spines than those obtained from the brain of wild-type mice. Therefore, we conclude that TRPM2 mediates the LPA-induced suppression of axonal growth, which provides a long-sought mechanism underlying the effect of LPA on neuronal development.

Anoctamin 1 contributes to inflammatory and nerve-injury induced hypersensitivity.

BACKGROUND: Various pathological conditions such as inflammation or injury can evoke pain hypersensitivity. That represents the response to innocuous stimuli or exaggerated response to noxious stimuli. The molecular mechanism based on the pain hypersensitivity is associated with changes in many of ion channels in dorsal-root ganglion (DRG) neurons. Anoctamin 1 (ANO1/TMEM16A), a Ca2+ activated chloride channel is highly visible in small DRG neurons and responds to heat. Mice with an abolished function of ANO1 in DRG neurons demonstrated attenuated pain-like behaviors when exposed to noxious heat, suggesting a role in acute thermal nociception. In this study, we further examined the function of ANO1 in mediating inflammation- or injury-induced hyperalgesia or allodynia. RESULTS: Using Advillin/Ano1fl/fl (Adv/Ano1fl/fl) mice that have a functional ablation of Ano1 mainly in DRG neurons, we were able to determine its role in mediating thermal hyperalgesia and mechanical allodynia induced by inflammation or nerve injury. The thermal hyperalgesia and mechanical allodynia induced by carrageenan injection and spared-nerve injury were significantly reduced in Adv/Ano1fl/fl mice. In addition, flinching or licking behavior after bradykinin or formalin injection was also significantly reduced in Adv/Ano1fl/fl mice. Since pathological conditions augment nociceptive behaviors, we expected ANO1's contribution to the excitability of DRG neurons. Indeed, the application of inflammatory mediators reduced the threshold for action potential (rheobase) or time for induction of the first action potential in DRG neurons isolated from control (Ano1fl/fl) mice. These parameters for neuronal excitability induced by inflammatory mediators were not changed in Adv/Ano1fl/fl mice, suggesting an active contribution of ANO1 in augmenting the neuronal excitability. CONCLUSIONS: In addition to ANO1's role in mediating acute thermal pain as a heat sensor, ANO1 is also capable of augmenting the excitability of DRG neurons under inflammatory or neuropathic conditions and thereby aggravates inflammation- or tissue injury-induced pathological pain.

TMEM16A confers receptor-activated calcium-dependent chloride conductance.

Calcium (Ca(2+))-activated chloride channels are fundamental mediators in numerous physiological processes including transepithelial secretion, cardiac and neuronal excitation, sensory transduction, smooth muscle contraction and fertilization. Despite their physiological importance, their molecular identity has remained largely unknown. Here we show that transmembrane protein 16A (TMEM16A, which we also call anoctamin 1 (ANO1)) is a bona fide Ca(2+)-activated chloride channel that is activated by intracellular Ca(2+) and Ca(2+)-mobilizing stimuli. With eight putative transmembrane domains and no apparent similarity to previously characterized channels, ANO1 defines a new family of ionic channels. The biophysical properties as well as the pharmacological profile of ANO1 are in full agreement with native Ca(2+)-activated chloride currents. ANO1 is expressed in various secretory epithelia, the retina and sensory neurons. Furthermore, knockdown of mouse Ano1 markedly reduced native Ca(2+)-activated chloride currents as well as saliva production in mice. We conclude that ANO1 is a candidate Ca(2+)-activated chloride channel that mediates receptor-activated chloride currents in diverse physiological processes.

Anoctamin 1, a multi-modal player in pain and itch.

Anoctamin 1 (ANO1/TMEM16A) encodes a Ca2+-activated Cl- channel. Among ANO1's many physiological functions, it plays a significant role in mediating nociception and itch. ANO1 is activated by intracellular Ca2+ and depolarization. Additionally, ANO1 is activated by heat above 44 °C, suggesting heat as another activation stimulus. ANO1 is highly expressed in nociceptors, indicating a role in nociception. Conditional Ano1 ablation in dorsal root ganglion (DRG) neurons results in a reduction in acute thermal pain, as well as thermal and mechanical allodynia or hyperalgesia evoked by inflammation or nerve injury. Pharmacological interventions also lead to a reduction in nocifensive behaviors. ANO1 is functionally linked to the bradykinin receptor and TRPV1. Bradykinin stimulates ANO1 via IP3-mediated Ca2+ release from intracellular stores, whereas TRPV1 stimulates ANO1 via a combination of Ca2+ influx and release. Nerve injury causes upregulation of ANO1 expression in DRG neurons, which is blocked by ANO1 antagonists. Due to its role in nociception, strong and specific ANO1 antagonists have been developed. ANO1 is also expressed in pruritoceptors, mediating Mas-related G protein-coupled receptors (Mrgprs)-dependent itch. The activation of ANO1 leads to chloride efflux and depolarization due to high intracellular chloride concentrations, causing pain and itch. Thus, ANO1 could be a potential target for the development of new drugs treating pain and itch.

Tentonin 3 is a pore-forming subunit of a slow inactivation mechanosensitive channel.

Mechanically activating (MA) channels transduce numerous physiological functions. Tentonin 3/TMEM150C (TTN3) confers MA currents with slow inactivation kinetics in somato- and barosensory neurons. However, questions were raised about its role as a Piezo1 regulator and its potential as a channel pore. Here, we demonstrate that purified TTN3 proteins incorporated into the lipid bilayer displayed spontaneous and pressure-sensitive channel currents. These MA currents were conserved across vertebrates and differ from Piezo1 in activation threshold and pharmacological response. Deep neural network structure prediction programs coupled with mutagenetic analysis predicted a rectangular-shaped, tetrameric structure with six transmembrane helices and a pore at the inter-subunit center. The putative pore aligned with two helices of each subunit and had constriction sites whose mutations changed the MA currents. These findings suggest that TTN3 is a pore-forming subunit of a distinct slow inactivation MA channel, potentially possessing a tetrameric structure.

Axonal neuropathy-associated TRPV4 regulates neurotrophic factor-derived axonal growth.

Spinal muscular atrophy and hereditary motor and sensory neuropathies are characterized by muscle weakness and atrophy caused by the degenerations of peripheral motor and sensory nerves. Recent advances in genetics have resulted in the identification of missense mutations in TRPV4 in patients with these hereditary neuropathies. Neurodegeneration caused by Ca(2+) overload due to the gain-of-function mutation of TRPV4 was suggested as the molecular mechanism for the neuropathies. Despite the importance of TRPV4 mutations in causing neuropathies, the precise role of TRPV4 in the sensory/motor neurons is unknown. Here, we report that TRPV4 mediates neurotrophic factor-derived neuritogenesis in developing peripheral neurons. TRPV4 was found to be highly expressed in sensory and spinal motor neurons in early development as well as in the adult, and the overexpression or chemical activation of TRPV4 was found to promote neuritogenesis in sensory neurons as well as PC12 cells, whereas its knockdown and pharmacologic inhibition had the opposite effect. More importantly, nerve growth factor or cAMP treatment up-regulated the expression of phospholipase A(2) and TRPV4. Neurotrophic factor-derived neuritogenesis appears to be regulated by the phospholipase A(2)-mediated TRPV4 pathway. These findings show that TRPV4 mediates neurotrophic factor-induced neuritogenesis in developing peripheral nerves. Because neurotrophic factors are essential for the maintenance of peripheral nerves, these findings suggest that aberrant TRPV4 activity may lead to some types of pathology of sensory and motor nerves.

Characterization of arginylation branch of N-end rule pathway in G-protein-mediated proliferation and signaling of cardiomyocytes.

The N-end rule pathway is a proteolytic system in which destabilizing N-terminal amino acids of short lived proteins are recognized by recognition components (N-recognins) as an essential element of degrons, called N-degrons. In eukaryotes, the major way to generate N-degrons is through arginylation by ATE1 arginyl-tRNA-protein transferases, which transfer Arg from aminoacyl-tRNA to N-terminal Asp and Glu (and Cys as well in mammals). We have shown previously that ATE1-deficient mice die during embryogenesis with defects in cardiac and vascular development. Here, we characterized the arginylation-dependent N-end rule pathway in cardiomyocytes. Our results suggest that the cardiac and vascular defects in ATE1-deficient embryos are independent from each other and cell-autonomous. ATE1-deficient myocardium and cardiomyocytes therein, but not non-cardiomyocytes, showed reduced DNA synthesis and mitotic activity ~24 h before the onset of cardiac and vascular defects at embryonic day 12.5 associated with the impairment in the phospholipase C/PKC-MEK1-ERK axis of Gα(q)-mediated cardiac signaling pathways. Cardiac overexpression of Gα(q) rescued ATE1-deficient embryos from thin myocardium and ventricular septal defect but not from vascular defects, genetically dissecting vascular defects from cardiac defects. The misregulation in cardiovascular signaling can be attributed in part to the failure in hypoxia-sensitive degradation of RGS4, a GTPase-activating protein for Gα(q). This study is the first to characterize the N-end rule pathway in cardiomyocytes and reveals the role of its arginylation branch in Gα(q)-mediated signaling of cardiomyocytes in part through N-degron-based, oxygen-sensitive proteolysis of G-protein regulators.

Voluntary movements as a possible non-reflexive pain assay.

BACKGROUND: The quantification of pain intensity in vivo is essential for identifying the mechanisms of various types of pain or for evaluating the effects of different analgesics. A variety of behavioral tests for pain measurement have been devised, but many are limited because animals are physically restricted, which affects pain sensation. In this study, pain assessment was attempted with minimal physical restriction, and voluntary movements of unrestrained animals were used to evaluate the intensities of various types of pain. RESULTS: The number of times animals reared or total distances traveled was measured using a motion-tracking device and found to be markedly reduced in carrageenan-induced inflammatory, acetic acid-induced visceral, and streptozotocin-induced neuropathic pain tests. These two voluntary movement parameters were found to be highly correlated with paw withdrawal latency from irradiating heat. In addition, these parameters were markedly reversed by morphine and by non-steroidal anti-inflammatory drugs in inflammatory pain models. These parameters were also useful to detect hypoalgesia in TRPV1⁻/⁻ mice. CONCLUSIONS: These results suggest that parameters of voluntary movement, such as, number of rearing and total distance moved, are effective indicators of pain intensity for many types of pain and that they can be used to evaluate degree of pain perception.

Anoctamin 1 mediates thermal pain as a heat sensor.

Vertebrates can sense and avoid noxious heat that evokes pain. Many thermoTRP channels are associated with temperature sensation. TRPV1 is a representative ion channel that is activated by noxious heat. Anoctamin 1 (ANO1) is a Cl- channel activated by calcium that is highly expressed in small sensory neurons, colocalized with markers for nociceptors, and most surprisingly, activated by noxious heat over 44oC. Although ANO1 is a Cl- channel, opening of this channel leads to depolarization of sensory neurons, suggesting a role in nociception. Indeed, the functional deletion of ANO1 in sensory neurons triggers the reduction in thermal pain sensation. Thus, it seems clear that ANO1 is a heat sensor in a nociceptive pathway. Since ANO1 modulators are developed for the purpose of treating chronic diseases such as cystic fibrosis, this finding is likely to predict unwanted effects and provide a guide for better developmental strategy.

Anoctamin 1/TMEM16A in pruritoceptors is essential for Mas-related G protein receptor-dependent itch.

ABSTRACT: Itch is an unpleasant sensation that evokes a desire to scratch. Pathologic conditions such as allergy or atopic dermatitis produce severe itching sensation. Mas-related G protein receptors (Mrgprs) are receptors for many endogenous pruritogens. However, signaling pathways downstream to these receptors in dorsal root ganglion (DRG) neurons are not yet understood. We found that anoctamin 1 (ANO1), a Ca 2+ -activated chloride channel, is a transduction channel mediating Mrgpr-dependent itch signals. Genetic ablation of Ano1 in DRG neurons displayed a significant reduction in scratching behaviors in response to acute and chronic Mrgpr-dependent itch models and the epidermal hyperplasia induced by dry skin. In vivo Ca 2+ imaging and electrophysiological recording revealed that chloroquine and other agonists of Mrgprs excited DRG neurons via ANO1. More importantly, the overexpression of Ano1 in DRG neurons of Ano1 -deficient mice rescued the impaired itching observed in Ano1 -deficient mice. These results demonstrate that ANO1 mediates the Mrgpr-dependent itch signaling in pruriceptors and provides clues to treating pathologic itch syndromes.

Lotus Seed Green Embryo Extract and a Purified Glycosyloxyflavone Constituent, Narcissoside, Activate TRPV1 Channels in Dorsal Root Ganglion Sensory Neurons.

Several studies have documented the broad-spectrum bioactivities of a lotus seed (Plumula nelumbinis [PN]) green embryo extract. However, the specific bioactive components and associated molecular mechanisms remain largely unknown. This study aimed to identify the ion channel-activating mechanisms of PN extracts. Using fluorometric imaging and patch-clamp recordings, PN extracts were screened for calcium channel activation in dorsal root ganglion (DRG) neurons. The TRPV1 channels in DRG neurons were strongly activated by the PN extract (mean amplitude of 131 ± 45 pA at 200 µg/mL) and its purified glycosyloxyflavone narcissoside (401 ± 271 pA at 100 µM). Serial treatment with a 200 µg/mL PN extract in TRPV1-overexpressing HEK293T cells induced robust desensitization to 10 ± 10% of the initial current amplitude. Thus, we propose that the PN extract and narcissoside function as TRPV1 agonists. This new finding may advance our knowledge regarding the traditional and scientific functions of PN in human health and disease.

Amplification of olfactory signals by Anoctamin 9 is important for mammalian olfaction.

Sensing smells of foods, prey, or predators determines animal survival. Olfactory sensory neurons in the olfactory epithelium (OE) detect odorants, where cAMP and Ca2+ play a significant role in transducing odorant inputs to electrical activity. Here we show Anoctamin 9, a cation channel activated by cAMP/PKA pathway, is expressed in the OE and amplifies olfactory signals. Ano9-deficient mice had reduced olfactory behavioral sensitivity, electro-olfactogram signals, and neural activity in the olfactory bulb. In line with the difference in olfaction between birds and other vertebrates, chick ANO9 failed to respond to odorants, whereas chick CNGA2, a major transduction channel, showed greater responses to cAMP. Thus, we concluded that the signal amplification by ANO9 is important for mammalian olfactory transduction.